Process for preparation of an agglutination reagent for rapid detection of typhoid

ABSTRACT

A process for the preparation of an agglutination reagent for rapid and early detection of typhoid, comprising the steps of: preparing  Salmonella typhi  specific antibody; preparing latex particles suspension; and coating said latex particles with the antibody.

FIELD OF INVENTION

The present invention relates to a process for the preparation of anagglutination reagent for rapid and early detection of Salmonella typhiin serum.

PRIOR ART

Typhoid is an endemic febrile disease caused by Salmonella typhi.Typhoid is a major concern of public health. The organism usually entersthe body by consumption of contaminated food or water and penetrates theintestinal wall. After that if multiplies and enters blood stream within24-72 hours resulting in enteric fever and bacteremia. After anincubation period of 10 to 14 days, early symptoms of typhoid, likeheadache, fever, loss of appetite, bradycardia, splenomegaly etc.appear. Typhoid is diagnosed either by blood culture or by detection ofits antigens or by the detection of its antibodies in the blood.

One of the most adapted methods for diagnosing the typhoid fever is theperformance of “Widal test”, a serological test based on the detectionof antibodies in the blood. This test is based on the fact thatantibodies against typhoid, remain in the blood of infected person, bindto the bacteria and results in the clumps formation which is referred as“Widal Agglutination”.

One of the imitation of the widal test is that the test is not specificas i cross reacts with other febrile organisms and many organisms offamily Enterobacteriaceae.

Another limitation of the widal test is that, as typhoid is an endemicdisease hence there always exist some background level of antibody inthe endemic areas. Hence it becomes necessary to determine the cut-offtitra for each region to rule out the possibility of diagnosis as falsepositive.

Yet another limitation of the widal test is that it gives positionresults only after one or two weeks of the onset of fever.

Still another limitation of the widal test is that test it is to beperformed on paired serum samples taken at an interval of at least oneweek apart because single widal test is elusive and inconclusive.

Further limitation of the widal test is that the antibioticadministration in the early phase of infection, inhibits the developmentof the antibody and hence test may give false negative result.

Still further limitation of the widal test is that TAB vaccinated normalhealthy persons give false positive reaction in widal test due topresence of circulating antibody against vaccine in human system.

Another limitation of the widal test is that it gives indirect evidenceof typhoid infection.

Further limitation of the widal test is that the test has lowsensitivity and low specificity.

Other technique known for diagnosis of typhoid is based upon isolationand identification of causative agent. This procedure is termed asgolden standard.

In this technique Salmonella typhi is isolated from blood and identifiedby microscopic and biochemical tests. However, this technique has manylimitations.

One limitation of the above technique is that it is time consuming as itrequires long period of incubation from 3 days to 14 days and alsorequires elaborate laboratory facilities.

Another limitation of the above technique is that for its performancelarge quantity of blood sample (10 ml/patient) is required.

Yet another limitation of the above technique is that it needs largevolume of culture medium i.e. 100 ml (10 times of blood sample).

Still another imitation of the above technique is its low sensitivity(40 to 60%), as there are very few organism in circulation, as low as1/ml which leads to false negative results.

Further limitation of above method is that bacterial growth in cultureis inhibited by serum bactericidal agents, present in blood which maylead to false negative results.

Still further limitation of blood culture is that antibiotics treatmentduring early phase of infection may inhibit bacterial growth in culturewhich may give false negative results.

Other known techniques such as Radioimmunoassay (RIA), Enzyme—linkedimmunosorbent assay etc. are based on detection of circulating antigenin the body fluids, but these techniques have many limitations.

One limitation of these techniques is that they require sophisticatedand elaborate laboratory facilities.

Another limitation of RIA is that K requires radioactive material whichis health hazard and also needs trained personnel to handle theradioactive material.

Still further limitation of above techniques is that reagents areexpensive.

Further limitation of these techniques is that minimum 4-5 hours arerequired to perform the tests.

OBJECT OF THE INVENTION

The primary object of the present invention is to provide a process forthe preparation of an agglutination reagent for rapid and earlydetection of S.typhi in serum samples of suspected typhoid patients.

Another object of the present invention is to provide a process for thepreparation of an agglutination reagent wherein the proposed reagentenables diagnosis of typhoid within 3 minutes after collection of serumsamples.

Yet another object of the present invention is to provide a process forthe preparation of an agglutination reagent wherein the serum samplerequired for the diagnosis of typhoid disease is as small as 20 μl.

Further object of the present invention is to provide a process for thepreparation of an agglutination reagent wherein the reagent enables thedetection of typhoid bacteria by simple latex agglutination technique.

Still further object of the present invention is to provide a processfor the preparation of an agglutination reagent wherein the reagentenables specific identification of Salmonella typhi antigen in serumsamples of suspected typhoid patients.

Yet further object of the present invention is to provide a process forthe preparation of an agglutination reagent wherein the reagent enablesthe diagnosis of typhoid in the early stages of infection even withinone or two days after the on set of the fever.

Another object of the present invention is to provide a process for thepreparation of an agglutination reagent wherein the reagent is highlysensitive.

Yet another object of the present mention is to provide a process forthe preparation of an agglutination reagent which enables diagnosis ofthe disease in field conditions as it does not require any equipment orlaboratory facility.

Still further object of the present invention is to provide a processfor the preparation of an agglutination reagent that does not requireany specially trained personnel to perform the test.

Yet further object of the present invention is to provide a process forthe preparation of an agglutination reagent which enables the diagnosisof those patients who have been administered with antibiotics resultingin blood culture isolation as negative.

DESCRIPTION OF PROCESS

According to the preferred embodiment of the present invention, theagglutination reagent is prepared by a process comprising of followingsteps:

(a) Preparation of Antibody (Immunoglobulins):

Flagellin geme sequence specific to Salmonella typhi is cloned andexpressed by recombinant DNA technology. The expressed recombinantprotein is purified by affinity chromatography. Hyper immune seraagainst this recombinant protein is raised in rabbit. Immunoglobulinfraction of hyper immune sera is separated by ammonium sulphateprecipitation. The precipitated immunoglobulins are suspended in 50 mMphosphate buffer (pH 7.2), dialysed and protein content determined.

(b) Preparation of Latex Particles Suspension:

1% carboxylated latex particles of size 0.88 to 0.90 μm and 40 mM 2-NMorphilinoethane sulphonic acid (MES) buffer (pH 5.5-6.0) are taken in apreferred ratio of 1:1 in a tube. They are mixed on a vortex mixer foraround 60 seconds and centrifuged at 10,000 rpm for 10-12 minutes atabout 4° C. The latex particles are further washed twice in 20 mM MESbuffer of pH 5.5 by mixing on vortex mixer for around 60 seconds,followed by centrifugation at 10,000 rmp for 10-12 minutes at about 4°C. After the final wash, the latex particles are suspended in 20 mM MESbuffer of pH 5.5 and the volume is made up equal to the starting volumeof the latex particles. The suspension is then sonicated by a tipsonicator at about 5 watts for 60-120 seconds, preferably 90 seconds. Tothis suspension freshly prepared solution of 0.1M1-ethyl-3(3-dimethyl-amino-propyl)carbodimide hydrochloride (EDC) in 20mM MES buffer (pH 5.5) taken in the preferred ratio of 1:1, is addeddrop wise, while the solution is slowly vortexed. The tube is rotatedslowly end-over-end for about 3 hours at a temperature of 20-25° C. Itis then washed thrice with 20 mM MES buffer (pH 5.5) at 10,000 rpm for10-12 minutes at a temperature of about 4° C. The latex particles areresuspended in MES buffer (20 mM, pH 5.5) and sonicated for 60-120seconds by a tip sonicator at 5 watts.

(c) Coating of Latex Particles with Antibody (Immunoglobulins):

To the suspension of latex particles prepared in step (b), 0.6-1.0 mgpreferably 0.8 mg per ml of the suspension, immunolobulins prepared instep (a) are added. The whole mixture is then rotated end-over-end for18-20 hours at a temperature of 20-25° C. The coating reaction isstopped by addition of 1M glycine (pH 11.0) taken in quantity of 0.06 mlper ml of solution of immunoglobulin coated latex particles. Rotation iscontinued for about 30 minutes at a temperature of 20-25° C. The coatedlatex particles are pelleted out by centrifugation at 10,000 rpm for10-12 minutes at a temperature of about 4° C. The pellet is washedthrice with washing buffer (50 mM glycine, pH 8.5; 0.03% triton X-100and 0.05% sodium azide) at 10,000 rpm for 10-12 minutes at a temperatureof about 4° C. Finally the washed coated latex particles are resuspendedin storage buffer (50 mM glycine, pH 8.5; 1.0% bovine serum albumin;0.03% triton X-100; 0.1% sodium azide and 0.01% thiomersol to a finalconcentration of 1% and sonicated by a tip sonicator for around 60seconds at about 5 watts and stored at 4° C.

METHOD OF USE

(a) Take 20-40 μl (1 to 2 drops) of test serum, positive and negativecontrols at three distinct places on a glass slide

(b) Add 10-2 μl (1-2 small drops) of latex reagent to test serum,positive and negative controls

(c) Mix the reactants with separate wooden sticks carefully to avoid anyintermixing of reactant placed at separate places and rotate the slidefor 1-2 minutes.

A positive reaction is indicated by the development of an agglutinationwithin 1-2 minutes of mixing the reagent with the test sample andpositive control, showing clearly visible clumping of the particles. Thespeed of appearance and quality of agglutination depends on the strengthof the antigen present, varying from large clumps which appear within afew seconds of mixing, to small clumps which develop rather slowly. Innegative reaction the reagent does not agglutinate and the cloudiness orthe turbid nature remains substantially unchanged throughout the lest.

Laboratory studies on the reliability of proposed agglutination reagentfor rapid detection of Salmonella typhi in typhoid patient serum isperformed with the laboratory strains of Salmonella typhi; and cultureproven and widal positive serum samples collected from suspected casesof typhoid; and with serum samples of apparently normal healthyindividuals. The result indicate 93.00% senshtWty and 98.00%specificity.

The present invention will now be illustrated with a working examplewhich is intended to be illustrative example and is not intended to betaken restrictively to imply any limitation on the scope of the presentinvention.

WORKING EXAMPLE

Flagellin gene sequence specific to salmonella typhi was amplified bypolymerase chain reaction (PCR) using gene specific primers. AmplifiedPCR product was cloned in Glutathione-S-transferase (GST) vector andlater expressed. The expressed protein was purified by GST affinitycolumn chromatography. The protein content of the purified product wasdetermined by Bradford method. Hyper immune serum against this proteinwas raised in rabbit. Immunoglobulins fraction of hyper immune sera wasseparated by ammonium sulphate precipitation. The precipitatedimmunoglobulins were suspended in 1.0 ml PB (50 mM, pH 7.2), dialysedand protein content determined. 1.0 ml of 1% carboxylated latexparticles and 1.0 ml of 40 mM MES buffer (pH 5.5-6.5) were taken in 2.0ml microcentrifuge tube. Then they were mixed on vortex mixer for 60seconds and centrifuged at 10,000 RPM for 10-12 minutes at a temperatureof 4° C. The latex particles were further washed twice in 2.0 ml of 20mM MES buffer (pH 5.5) by mixing on vortex mixer for 60 seconds andcentrifugation at 10,000 RPM for 10-12 minutes at a temperature of 4° C.Following the final wash, the latex particles were suspended in 1.0 mlMES buffer (20 mM, pH 5.5) and sonicated by a tip sonicator at 5 wattsfor 60-120 seconds. Later 1.0 ml of freshly prepared solution of 0.1 M1-ethyl-3-(3-dimethylaminopropyl)carbodimide hydrochloride (EDC) in MESbuffer (20 mM, pH 5.5) was added drop wise while the solution was slowlyvortexed. Then the tube was rotated slowly end-over-end for 3 hours at atemperature of 20-25° C. followed by washing three times with MES buffer(20 mM, pH 5.5) at 10,000 RPM for 10-12 minutes at a temperature of 4°C. The latex particles were resuspended in 0.7 ml MES buffer (20 mM, pH5.5) and sonicated for 60-120 seconds by a tip sonicator at 5 watts. 0.8mg of immunoglobulins were added to latex particles and volume was madeup to 1.0 ml with MES buffer (20 mM, pH 5.5). This was then rotatedend-over-end for 18-20 hours at a temperature of 20-25° C. The coatingreaction was then stopped by addition of 0.06 ml of 1M glycine (pH11.0). The rotation was continued for 30 minutes at a temperature of20-25° C. The coated latex particles were pelleted out by centrifugationat 10,000 RPM for 10-12 minutes at a temperature of 4° C. The pellet waswashed thrice with 2.0 ml of washing buffer (50 mM glycine, pH 8.5,0.03% triton X-100 and 0.05% sodium azide) at 10,000 RPM for 10-12minutes at a temperature of 4° C. The washed coated latex particles wereresuspended in storage buffer (50 mM glycine, pH 8.6. 1.0% bovine serumalbumin, 0.03% triton X-100, 0.1% sodium azide and 0.01% thiomersol) toa final concentration of 1% and sonicated with tip sonicator for 60seconds at 5 watts and stored at 4° C.

It is to be understood that the present invention is susceptible tomodifications, changes and adaptations by those skilled in the art. Suchmodifications, changes, adaptations are intended to be within the scopeof the present invention which is further set forth under the followingclaims:

1-22. (canceled)
 23. A process for the preparation of an agglutinationreagent for rapid and early detection of typhoid comprising: (a)preparing antibody specific to Salmonella typhi; (b) preparing latexparticles suspension; (c) coating of the said latex particles with thesaid antibody; wherein the said process of preparing antibody specificto Salmonella typhi comprises cloning Flagellin gene sequence specificto Salmonella typhi, expressing the said Flagellin gene sequence byrecombinant DNA technology, followed by purifying recombinant protein byaffinity chromatography, raising the hyper immune sera against purifiedrecombinant protein in animals like rabbit, separating the antibody(immunoglobulin) fraction of hyper immune sera by precipitating inammonium sulphate, suspending in 50 mM phosphate buffer of pH 7.2 anddialyzing; wherein the said process of preparing latex particlesuspension comprises: (i) mixing 1% carboxylated latex particles of size0.88 to 0.90 μm and 40 mM 2-N Morphilinoethane sulphonic acid (MES)buffer of pH 5.5 to 6.0 in a ratio of 1:1 on a vortex mixer for about 60seconds, centrifuging at 10,000 rpm for 10-12 minutes at about 4° C.,followed by washing twice with 20 mM MES buffer of pH 5.5 at 10,000 rpmfor 10-12 minutes at about 4° C., sonicating by a tip sonicator at about5 watts for 60-120 seconds; (ii) adding drop wise a freshly preparedsolution of 0.1 M 1-ethyl-3 (3-dimethyl-amino propyl)carbodimidehydrochloride (EDC) in 20 mM MES buffer of pH 5.5 to the said sonicatedlatex particles obtained from step (i) above in a ratio of 1:1 whilevortexing the suspension slowly, rotating the suspension slowlyend-over-end for about 3 hours at a temperature of 20-25° C., washingthrice with 20 mM MES buffer (pH 5.5) followed by sonicating the washedsuspension of latex particles by a tip sonicator for 60-120 seconds atabout 5 watts; wherein the said process of coating of the said latexparticles is done by adding 0.6-1.0 mg preferably 0.8 mg per ml of thesaid antibody (immunoglobulins) to the said latex particle suspension,rotating the suspension end-over-end for 18-20 hours at a temperature ofabout 20-25° C., stopping the coating reaction by 1M glycine (pH 11.0)taken in quantity of 0.06 ml per ml of solution of antibody coated latexparticles followed by centrifugation at 10,000 rpm for 10-12 minutes ata temperature of about 4° C., washing thrice with washing buffercomprised of 50 mM glycine, pH 8.5; 0.03% triton X-100 and 0.05% sodiumazide, suspending in storage buffer to a final concentration of 1%,sonicating for about 60 seconds at about 5 watts and storing at 4° C.24. An agglutination reagent for rapid and early detection of typhoid,comprising of 1% carboxylated latex particles coated with antibodyspecific to Salmonella typhi, suspended in storage buffer.
 25. Theagglutination reagent as claimed in claim 24, wherein the size of thesaid latex particles is 0.88 to 0.90 μm.
 26. The agglutination reagentas claimed in claim 24, wherein the said storage buffer is comprised of50 mM glycine pH 8.5, 1.0% bovine serum albumin, 0.03% triton X-100,0.1% sodium azide and 0.01% thiomersal.
 27. The agglutination reagentfor rapid and early detection of typhoid as claimed in claim 24, whereinthe said antibody is the immunoglobulin fraction, of the hyper immunesera raised in rabbit against the recombinant protein expressed bycloning of Flagellin gene sequence specific to Salmonella typhi byrecombinant DNA technology, suspended in 50 mM phosphate buffer.
 28. Akit for rapid and early detection of typhoid comprising 1% agglutinationreagent as claimed in claim 24 suspended in storage buffer, glassslides, droppers, wooden sticks and positive and negative controls.